Bacterial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) in Polluted Estuarine Sediments of the Lagos Lagoon, Nigeria

Author: Obi, Chioma Christiana

Supervisors: Amund O. O. and Ugoji E. O.

potential effects of these pollutants on estuaries. In this study, sediment samples were collected from three sites namely; Apapa, Ofin and Eledu in the Lagos Lagoon. Levels of polycyclic aromatic hydrocarbons (PAHs) and heavy metal contamination of these sites were quantified using GC-FID and flame ionization atomic absorption spectrophotometry respectively. Continual aerobic enrichment procedure with pyrene, fluoranthene, fluorene, benz(a)anthracene supplied as the sole carbon was used in the isolation of bacterial strains that were able to utilize mixed PAHs. The degradation abilities of the isolates and the detection of metabolic intermediates were determined using HPLC HP 5890 series and GC-MS (HP 6890 series) respectively. The isolates were identified by the amplification and sequencing of their 16S rRNA gene. Microbial community structures of the sites were investigated using Illumina sequencing technique. Amplification of the polycyclic aromatic ring hydroxylating dioxygenase (PAHRHD) gene catalyzing the initial hydroxylation of the aromatic ring during PAHs biodegradation was carried out using NidA-forward/NidA-reverse and FRT6A/FRT4B. Protein expression pattern of the isolates in the presence of PAHs was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results from the determination of physico-chemical parameters showed that Apapa sampling point had the highest level of total PAHs and heavy metal contamination (5.6 μg/kg, 71.90 mg/kg) followed by Ofin (3947 μg/kg, 67 mg/kg) and Eledu (2991 μg/kg, 54.62mg/kg) respectively. Selective aerobic enrichment procedure with pyrene, benz(a)anthracene, fluorene, fluoranthene as sole sources of carbon and energy yielded 12 bacterial isolates. Two strains exhibiting broad spectrum PAH utilization with production of metabolic intermediates were selected for further studies. These were identified as Pseudomonas aeruginosa strain F4b and Rhodococcus sp. strain FF2 with GenBank accession numbers KT894767 and KT894768 respectively. Strain F4b tolerated up to 6.0 mM of copper while strain FF2 was able to tolerate only 3.0 mM of copper. Both strains showed resistance to ampicillin (25 μg), Erythromycin (10 μg), penicillin (10 μg) and cloxacillin (5 μg). The biodegradation studies carried out with strains FF2 and F4b showed rapid exponential increase in cell numbers on some PAHs with average of 86% substrate utilization. Both strains produced metabolic intermediates including benz(a)anthracene 7, 12 dione, 9H Fluorene-9-one and 2,5 dihydroxybenzoic acid among others from benz(a)anthracene, fluorene and pyrene respectively during their growth. Microbial community structure of the sediment samples generated across all libraries a total of 565 bacterial operational taxonomic units (OTUs) (97 % of sequence reads) and 17 archaeal OTUs (3 % of sequence reads). Both the Shannon’s diversity (species richness) and Chao1 (species evenness) indices showed that the Ofin sediment had the highest diversity (9.04) followed by Apapa (5.2) and Eledu (3.99). Samples were dominated by a single OTU assigned to the family Helicobacteraceae (Epsilonproteobacteria). All Illumina sequences from the community studies have been deposited in the NCBI Sequence Read Archive under accession number SRP069095. Protein expression pattern in the presence of different PAHs using SDS-PAGE revealed that these PAHs induced some of the enzymes with molecular weight of 37 kDa that are involved directly or indirectly in the breakdown of these compounds. PAH-RHD genes amplified from both isolates were NarAa and NahAC genes for strain FF2 and F4b respectively. Degradation of the PAH mixtures during microcosm studies resulted in the removal of 40-80% of the total PAHs by indigenous microflora. Illumina sequencing of the 16S rRNA gene sequences generated across all libraries from the microcosm studies generated a total of 75,000 gene sequences which were clustered into 1229 OTUs (at the 97 % identity level). Proteobacteria was identified as the predominant phylum for all samples. PAH-RHD gene (Nid A type) from Mycobacterium sp. HH2 appeared to be the dominant gene followed by (2Fe-2S)-binding protein of Burkholderia sp. Ch1-1 and Rieske (2Fe-2S) protein from Marinobacterium sp. AK27 (Ndo type). This xxiii study has provided the evidence for the existence of an active aerobic autochthonous microbialcommunity in the coastal areas of the Lagos Lagoon with PAH metabolic functionalities.